Artemisinin (AN), produced by the plant Artemisia annua L., is a proven therapeutic for treating malaria and also for treatment of a large range of bacterial, viral and parasitic diseases, and cancers. While AN has high value as a therapeutic, its production is only cost effective when harvested from field grown plants. Understanding biosynthetic control of this important terpenoid drug is crucial to meeting the growing worldwide demand. Recently we showed that plants subjected to reactive oxygen stress produce higher levels of AN and its precursors. Since this phenomenon has the potential to link a variety of established AN elicitors in a unified model, we propose investigating reactive oxygen stress as a key regulator of AN biosynthesis. Our 3 objectives are: 1. Investigation of the relative expression levels of early (HMGR, FPS) and late dedicated (ADS, CYP71AV1, DBR2, ALDH1) genes as plants are subjected to diverse sources of oxidative stress, including photo, drought, and chemically induced oxidation. These data would serve to identify a transcriptional basis for ROS induction of AN production. 2. Compare metabolic profiles and the redox state of plants under normal and oxygen stressed growing conditions. Measuring key metabolic intermediates allows us to form a preliminary metabolic balance map showing key points of change upon ROS-induced perturbations to help further elucidate the ROS role in the putative non-enzymatic last step of AN biosynthesis and metabolite balance control not readily identifiable through transcript analysis. 3. Investigate and demonstrate in soil-grown plants a process for increasing AN production through the induction of oxidative stress by both biotic and abiotic means that will allow for optimizing production of AN while simultaneously allowing for maximum plant growth. This will further correlate potentially simple methods that could be employed immediately to induce mild oxidative stress for over production of artemisinin in soil-grown plants. These results will further our fundamental understanding of the biosynthesis of artemisinin in A. annua and will further our ability to enhance its production to meet the ever growing therapeutic demand. PUBLIC HEALTH RELEVANCE: Malaria and the neglected tropical diseases addressed in our proposal account for >500 million infections and upwards of 2 million deaths. Artemisinin has been shown to be effective against all of these diseases, but the drug is in desperately short supply to treat even malaria. Our studies on reactive oxygen will facilitate a better understanding of the last steps in the biosynthetic pathway thereby enhancing the potential to increase the drug produced in the plant.